title: Quantitative morphological analysis of arrestin2 clustering upon G protein-coupled receptor stimulation by super-resolution microscopy.
abstract: Clustering of arrestins upon G protein-coupled receptor stimulation is a phenomenon that is well-known but difficult to describe quantitatively due to the size of the clusters close to the diffraction limit of visible light. We introduce a general method to quantitatively investigate the clustering of arrestin following stimulation of the C-C chemokine receptor 5 (CCR5) using single-molecule super-resolution imaging and coordinate and image-based cluster analysis. We investigated the effect of potent anti-HIV ligands of CCR5 with different pharmacological profiles on arrestin2 cluster formation and found that only the ligands capable of inducing CCR5 internalization induced arrestin2 recruitment and clustering. We further demonstrate that the fraction of arrestin2 molecules found in clusters larger than 100nm correlates with the magnitude of ligand-induced CCR5 internalization, but not with G protein activation, indicating that recruitment of arrestin2 to CCR5 is independent of G protein activation. Pre-treatment of the cells with the drug cytochalasin D, which blocks actin polymerization, led to the formation of larger clusters, whereas the inhibitor of microtubule polymerization nocodazole had little effect on arrestin2 recruitment, suggesting an active role of actin in the organization and dynamics of these aggregates.
authors.affiliation: Department of Human Protein Sciences, University of Geneva, CMU, Rue Michel-Servet 1, 1211 Genève 4, Switzerland.
authors.forename: Zinnia
authors.initials: Z
authors.lastname: Truan
authors.forename: Laura
authors.initials: L
authors.lastname: Tarancón Díez
authors.forename: Claudia
authors.initials: C
authors.lastname: Bönsch
authors.forename: Sebastian
authors.initials: S
authors.lastname: Malkusch
authors.forename: Ulrike
authors.initials: U
authors.lastname: Endesfelder
authors.forename: Mihaela
authors.initials: M
authors.lastname: Munteanu
authors.forename: Oliver
authors.initials: O
authors.lastname: Hartley
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.forename: Alexandre
authors.initials: A
authors.lastname: Fürstenberg
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D016466:CHO Cells; D002417:Cattle; D018946:Chemokine CCL5 / Q000494:pharmacology / Q000502:physiology*; D019742:Chemokines, CC / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D015638:Cytochalasin D / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015739:Nocodazole / Q000494:pharmacology; D021381:Protein Transport; D019713:Receptors, CCR5 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000737:chemistry; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: C539265:5P12-RANTES; D019390:Arrestins; D018946:Chemokine CCL5; D019742:Chemokines, CC; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D050257:Tubulin Modulators; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D015739:Nocodazole
keywords: Arrestin; Clustering; G-protein coupled receptor; Single-molecule fluorescence microscopy; Super-resolution microscopy